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Stable Folding Core in the Folding Transition State of an Α-Helical Integral Membrane Protein

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Stable Folding Core in the Folding Transition State of an Α-Helical Integral Membrane Protein Stable folding core in the folding transition state of an α-helical integral membrane protein Paul Curnow1, Natalie D. Di Bartolo, Kathleen M. Moreton, Oluseye O. Ajoje, Nicholas P. Saggese, and Paula J. Booth1 School of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom Edited by Alan R. Fersht, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom, and approved July 12, 2011 (received for review August 26, 2010) Defining the structural features of a transition state is important to changes in the chromophore absorbance spectra, providing in understanding a folding reaction. Here, we use Φ-value and a sensitive intrinsic structural probe that can be used to follow double mutant analyses to probe the folding transition state of folding behavior. the membrane protein bacteriorhodopsin. We focus on the final bR can be reversibly unfolded according to a two-state reaction C-terminal helix, helix G, of this seven transmembrane helical in mixed lipid/detergent micelles (7–9) analogous to the unfold- protein. Φ-values could be derived for 12 amino acid residues in ing of aqueous proteins in urea or guanidinium. We recently used helix G, most of which have low or intermediate values, suggesting this folding system to study a series of Ala mutations through that native structure is disrupted at these amino acid positions in transmembrane helix B (9) and carried out a classical Φ-value the transition state. Notably, a cluster of residues between E204 analysis in order to obtain information on the nature of the fold- and M209 all have Φ-values close to zero. Disruption of helix G ing transition state (10). Φ-value analysis emerged from the work is further confirmed by a low Φ-value of 0.2 between residues of Fersht and colleagues as a method of analyzing the nature of T170 on helix F and S226 on helix G, suggesting the absence of the folding transition state at the level of individual amino acid a native hydrogen bond between helices F and G. Φ-values for residues (11–14). The method uses site-directed mutagenesis to paired mutations involved in four interhelical hydrogen bonds remove specific side-chain interactions and combines kinetic revealed that all but one of these bonds is absent in the transition and equilibrium data to assess the energetic penalty of removing Φ ΔΔ H2O state. The unstructured helix G contrasts with -values along helix these interactions from the folded state (given by Gu ) and BIOPHYSICS AND B that are generally high, implying native structure in helix B in the the transition state (ΔΔG ). The Φ-value is the ratio of TS-U COMPUTATIONAL BIOLOGY transition state. Thus helix B seems to constitute part of a stable these two terms (SI Appendix :Eq. S11). The Φ-value method folding nucleus while the consolidation of helix G is a relatively late is applied only if the protein is destabilized and ideally when H2O −1 −1 folding event. Polarization of secondary structure correlates with ΔΔGu is ≥0.6 kcal·mol and ≤1.7 kcal·mol (14), although sequence position, with a structured helix B near the N terminus more strongly destabilizing mutants are commonly used. Under contrasting with an unstructured C-terminal helix G. these conditions, if the transition state is not destabilized by the ΔΔ ¼ 0 Φ ¼ 0 mutation then GTS-U and thus . Because of the ntegral membrane proteins play a central role in cellular pro- equivalence between protein energetics and protein structure, Icesses such as signal transduction, solute transport, energy it is inferred that the mutated residue does not make native-like generation, and membrane-associated biochemistry. Membrane interactions in the transition state. If, however, the mutated re- proteins constitute ∼25% of the global proteome (1, 2) and sidue does make native-like contacts in the transition state then ΔΔ ¼ ΔΔ H2O Φ ¼ 1 Φ are distinguished by having a proportion of their polypeptide se- GTS-U Gu and . -values are thus a measure of quence buried within a lipid bilayer. These proteins are well the extent of structural consolidation at any specific site in the adapted to cope with the complex bilayer environment in which transition state. This rationale is sound for the “ideal” case of they reside. The most obvious adaptation is the strong sequence Φ-values of 0 and 1, but intermediate values are more ambiguous bias toward hydrophobic amino acids that can be accommodated (because they do not necessarily imply intermediate structure) in the low dielectric of the bilayer interior. The sequence diver- and must be interpreted with care. Φ-value analysis has been gence between membrane and aqueous proteins raises important widely used to characterize the transition states of a number of questions as to whether the forces that determine the structure different aqueous proteins [e.g., (11, 15–18)] and has provided and dynamics of aqueous proteins play equally important roles important mechanistic insight (19–21). However, Φ-value analy- in membrane proteins (3). Understanding these factors will be sis has not been widely used to study membrane protein folding particularly important for optimizing the conditions under which with only one example for an α-helical protein (10) and one for a membrane proteins are best expressed, solubilized, and reconsti- β-barrel protein (22). tuted for study in vitro; these steps are currently not trivial for We previously obtained Φ-values for seven residues in helix B most membrane proteins. of bR (10) and the results suggested that much of helix B has One area of current interest is membrane protein folding native-like structure in the transition state, with the exception of (4–6). Understanding the transition between folded and unfolded a region at the cytoplasmic end facing the interior of the protein. states will provide important information on folding mechanisms We also found that a global measure of the transition state struc- and on the core residues and interactions that control membrane ture, the β-value, was very low, suggesting that overall the transi- protein folding and stability. The archetypal membrane protein tion state is close to the partly structured, SDS-unfolded state bacteriorhodopsin (bR) has served well as a prototype for devel- oping methods that can be used to study the folding of the domi- Author contributions: P.C. and P.J.B. designed research; P.C., N.D.D.B., K.M.M., O.O.A., and nant structural class of alpha-helical membrane proteins. bR is N.P.S. performed research; P.C., N.D.D.B., O.O.A., N.P.S., and P.J.B. analyzed data; and P.C. a light-activated proton pump from the archae Halobacterium and P.J.B. wrote the paper. salinarum that consists of seven transmembrane alpha helices The authors declare no conflict of interest. denoted A-G according to their position in the amino acid se- This article is a PNAS Direct Submission. quence from N to C terminus. A key component of bR is the 1To whom correspondence may be addressed. E-mail: [email protected] or covalently bound retinal cofactor, which is essential for proton [email protected]. transport and generates a characteristic purple chromophore. This article contains supporting information online at www.pnas.org/lookup/suppl/ Changes to the structure of the retinal binding pocket give rise doi:10.1073/pnas.1012594108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1012594108 PNAS ∣ August 23, 2011 ∣ vol. 108 ∣ no. 34 ∣ 14133–14138 Downloaded by guest on September 24, 2021 Fig. 1. Equilibrium unfolding of bR. (A) Typical equi- librium curves from absorbance measurements show- ing changes in the slope and midpoint of the major unfolding transition for WT and two representative mutants E204A and M209A. (B) Mutants L207A, F219A, and L224A showed atypical equilibrium curves that could not be fit to a two-state equation. Following Faham et al., (9) the mutations in helix G were mapped onto the native state structure (C and D) and categorized according to the magni- H2O tude of ΔΔGu . Severely destabilizing mutations (red) were only found for residues that faced toward the protein interior, while sites of moderately desta- bilizing mutations (orange) faced other helices and the exterior. Mutations (blue) causing minor changes H2O in ΔΔGu faced the exterior. Mutation L201A, shown in yellow in (D), caused an increase in stability of 0.7 kcal·mol−1.(C and D) constructed in Pymol (48) using PDB file 1C3W (49). (the latter possessing over half the native helix content). A simple concentration is given throughout as a mole fraction of the total χ interpretation of these results leads to the hypothesis that the lipid and detergent, SDS, because the concentration of SDS bR transition state will comprise a stable folding core, which experienced by bR within the micelle will not be equivalent to minimally includes helix B. Here, we investigate whether this pro- the bulk concentration (31). The loss of the retinal chromophore, posed folding core contains elements of another helix of bR, by which has a characteristic absorbance band at 560 nm, was used extending Φ-value analysis to another transmembrane segment of as a folding probe. Circular dichroism measurements reveal that bR, the C-terminal helix G. This helix is of interest because it changes in retinal absorbance during SDS-unfolding are equiva- contains K216, the covalent attachment site for the retinal cofac- lent to a moderate reduction in secondary structure content tor, and previous data suggested that unlike helix B, helix G in from 74% to 42% associated with unfolding (8, 32, 33). The SDS- isolation is not inherently stable (23, 24) and that it folds later unfolded state of bR thus retains a considerable amount of sec- in the folding pathway in vitro (25–27) and in vivo (28).
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